Osteogenic differentiation of muscle precursor cells: involvement of the DNA Damage Response

Heterotopic ossification (HO) is a condition characterized by the deposition of ectopic mineralization in soft tissues. The causes of this phenomenon are still unclear but recent advances have shed light on the possibility that acute or chronic inflammation could trigger the aberrant differentiation of mesenchymal stem cells (MSCs). Among the soft tissues of the human body, skeletal muscle (SkM) is the one with the most relevant function for the maintenance of movement capability, postural behavior and metabolic homeostasis. SkM is frequently a site for the insurgence of HO, often in cases of traumatic injuries, accidents, surgery and paralysis. Given the high regeneration ability, SkM is a great source of MSCs that contribute to tissue homeostasis both in physiological and pathological conditions. Mesoangioblasts (MABs) are perivascular stem cells capable to differentiate in many mesodermal cell types as skeletal muscle, smooth muscle, adipocytes and osteoblasts. MABs have been extensively studied given their high migration and repair ability. Moreover, MABs are highly proliferative and maintain their stemness in vitro for a high number of passages. To investigate the signaling pathways controlling MAB differentiation trajectories, we performed a High-Content Screening (HCS) looking for substances that could modulate differentiation decisions. We treated MABs with 560 molecules from the Prestwick Chemicals Library to assess their myogenic and osteogenic differentiation upon pharmacological perturbation. For this purpose, we monitored the expression of the myogenic transcription factor myogenin (MYOG) and the osteogenic enzyme alkaline phosphatase (ALP), essential for the synthesis of the bone mineralized matrix. Among the four hits identified in the primary screening, only Idoxuridine (IdU) was shown to be able to trigger MAB osteogenic differentiation even in the absence of the osteogenic cytokine BMP-2. IdU is an iodinated thymidine analogue. It was extensively used as an anti-viral drug and radiotherapy adjuvant given its ability to counteract the DNA synthesis both of Herpes simplex virus and cancer cells. Its mechanism of action relies on the ability to be incorporated into the DNA double-helix, producing double strand breaks (DSBs). In the present work, I have investigated the mechanism of action of IdU in the MAB osteogenic conversion. IdU limits spontaneous MAB myogenesis by inhibiting the expression of MYOG while inducing the expression of ALP at early times. ALP expression is also paralleled by the induction of the osteogenic transcription factors RUNX2 and SP7. Moreover, given its ability to interact with the genomic DNA, IdU treatment results in a delay of MAB cell cycle progression. Furthermore, competition of IdU with the nucleotide thymidine, strongly limits the induction of osteogenic differentiation. Finally, IdU can induce DSBs on MAB DNA, triggering the signaling axis of the DNA Damage Response (DDR). In such cascade, the activity of the ATM kinase and p38-MAPK, two important actors in the DDR, were proven to be essential for the osteogenic induction. Overall, this thesis sheds light on the importance of this stress-responsive pathways in the regulation of the differentiation fate of muscle precursor stem cells.